Cyclohexenyl-ethyl-thiourea compounds and use

ABSTRACT

Novel CycloHexenyl-Ethyl-Thiourea (CHET) compounds as inhibitors of reverse transcriptase and effective agents for the treatment of HIV infection, including mutant, drug-sensitive, drug-resistant, and multi-drug resistant strains of HIV.

FIELD OF THE INVENTION

[0001] The invention relates to inhibitors of reverse transcriptaseeffective against HIV, including mutant strains of HIV, and effective inthe treatment of multi-drug resistant HIV infection.

BACKGROUND OF THE INVENTION

[0002] Agents currently used to treat HIV infection attempt to blockreplication of the HIV virus by blocking HIV reverse transcriptase or byblocking HIV protease. Three categories of anti-retroviral agents inclinical use are nucleoside analogs (such as AZT), protease inhibitors(such as nelfinavir), and the recently introduced non-nucleoside reversetranscriptase inhibitors (NNI), such as nevirapine.

[0003] The recent development of potent combination anti-retroviralregimens has significantly improved prognosis for persons with HIV andAIDS. Combination therapies may be a significant factor in the dramaticdecrease in deaths from AIDS (a decrease in death rate as well asabsolute number). The most commonly used combinations include twonucleoside analogs with or without a protease inhibitor.

[0004] Nevirapine is currently the only NNI compound which has been usedin combination with AZT and/or protease inhibitors for the treatment ofHIV. A new series of effective drug cocktails will most likely involveother NNIs in combination with nucleoside and protease inhibitors as atriple action treatment to combat the growing problem of drug resistanceencountered in single drug treatment strategies.

[0005] The high replication rate of the virus unfortunately leads togenetic variants (mutants), especially when selective pressure isintroduced in the form of drug treatment. These mutants are resistant tothe anti-viral agents previously administered to the patient. Switchingagents or using combination therapies may decrease or delay resistance,but because viral replication is not completely suppressed in singledrug treatment or even with a two drug combination, drug-resistant viralstrains ultimately emerge. Triple drug combinations employing one (ortwo) nucleoside analogs and two (or one) NNI targeting RT provide a verypromising therapy to overcome the drug resistance problem. RT mutantstrains resistant to such a triple action drug combination would mostlikely not be able to function.

[0006] Dozens of mutant strains have been characterized as resistant toNNI compounds, including L1001, K103N, V106A, E138K, Y181C and Y188H. Inparticular, the Y181C and K103N mutants may be the most difficult totreat, because they are resistant to most of the NNI compounds that havebeen examined.

[0007] Recently, a proposed strategy using a knock-out concentration ofNNI demonstrated very promising results. The key idea in this strategyis to administer a high concentration of NNI in the very beginningstages of treatment to reduce the virus to undetectable levels in orderto prevent the emergence of drug-resistant strains. The ideal NNIcompound for optimal use in this strategy and in a triple actioncombination must meet three criteria:

[0008] 1) very low cytotoxicity so it can be applied in high doses;

[0009] 2) very high potency so it can completely shut down viralreplication machinery before the virus has time to develop resistantmutant strains; and

[0010] 3) robust anti-viral activity against current clinically observeddrug resistant mutant strains.

[0011] Novel NNI designs able to reduce RT inhibition to subnanomolarconcentrations with improved robustness against the most commonlyobserved mutants and preferably able to inhibit the most troublesomemutants are urgently needed. New antiviral drugs will ideally have thefollowing desired characteristics: (1) potent inhibition of RT; (2)minimum cytotoxicity; and (3) improved ability to inhibit known,drug-resistant strains of HIV. Currently, few anti-HIV agents possessall of these desired properties.

[0012] Two non-nucleoside inhibitors (NNI) of HIV RT that have beenapproved by the U.S. Food and Drug Administration for licensing and salein the United States are nevirapine (dipyridodiazepinone derivative) anddelavirdine (bis(heteroaryl)piperazine (BHAP) derivative, BHAP U-90152).Other promising new non-nucleoside inhibitors (NNIS) that have beendeveloped to inhibit HIV RT include dihydroalkoxybenzyloxopyrimidine(DABO) derivatives, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine(HEPT) derivatives, CHETrahydrobenzondiazepine (TIBO),2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-spiro-5″-(4″-amino-1′,2″-oxathiole-2″,2′-dioxide)pyrimidine (TSAO), oxathiin carboxanilidederivatives, quinoxaline derivatives, thiadiazole derivatives, andphenethylthiazolylthiourea (PETT) derivatives.

[0013] NNIs have been found to bind to a specific allosteric site ofHIV-RT near the polymerase site and interfere with reverse transcriptionby altering either the conformation or mobility of RT, thereby leadingto a noncompetitive inhibition of the enzyme (Kohlstaedt, L. A. et al.,Science, 1992, 256, 1783-1790).

[0014] A number of crystal structures of RT complexed with NNIs havebeen reported (including α-APA, TIBO, Nevirapine, and HEPT derivatives),and such structural information provides the basis for furtherderivatization of NNI aimed at maximizing binding affinity to RT.However, the number of available crystal structures of RT NNI complexesis limited.

[0015] Given the lack of structural information, alternate designprocedures must be relied upon for preparing active inhibitors such asPETT and DABO derivatives. One of the first reported strategies forsystematic synthesis of PETT derivatives was the analysis ofstructure-activity relationships independent of the structuralproperties of RT and led to the development of some PETT derivativeswith significant anti-HIV activity (Bell, F. W. et al., J. Med. Chem.,1995, 38, 4929-4936; Cantrell, A. S. et al., J. Med. Chem., 1996, 39,4261-4274).

[0016] A series of selected phenethylthiazolylthiourea (PETT)derivatives targeting the NNI binding site of HIV reverse transcriptase(RT) were synthesized and tested for anti-human immunodeficiency virus(HIV) activity. The structure based design and synthesis of these PETTderivatives were aided by biological assays and their anti-HIV activity.Some of these novel derivatives were more active than AZT or Troviridineand abrogated HIV replication at nanomolar concentrations without anyevidence of cytotoxicity. These compounds are useful in the treatment ofHIV infection, and have particular efficacy against mutant strains,making them useful in the treatment of multi-drug resistant HIV.

SUMMARY OF THE INVENTION

[0017] The invention provides cyclohexenyl-ethyl-thiourea (CHET)compounds as newly identified non-nucleoside inhibitors (NNI) of HIVreverse transcriptase. The CHET compounds, compositions, and methods ofthe invention are useful in the treatment of HIV infection, withparticular efficacy against multiple strains of HIV, includingmulti-drug resistant mutant strains.

[0018] The CHET compounds, compositions, and methods of the inventionare useful for inhibiting reverse transcriptase activity and inhibitingreplication of multiple strains of HIV, including therapy-naive,drug-resistant, and multi-drug resistant strains. In particular, theCHET compounds of the invention are useful for treating retroviralinfection in a subject, such as an HIV-1 infection, by administration ofthe CHET compounds of the invention, for example, in a pharmaceuticalcomposition.

[0019] The CHET compounds of the invention contain a cyclohexnestructure as shown in Formula I. The cyclohexene may be substituted orunsubstituted (R_(n)). R₁ is a cyclic moiety which may be substituted orunsubstituted (X). The cyclic moiety can be aromatic and/orheterocyclic. X can be H, halo, nitro, or CF₃. X is preferably halo, andmost preferably is Br or Cl. In a preferred embodiment, R₁ is pyridinyl,preferably substituted (X) with Br or Cl. Exemplary CHET compounds ofthe invention are HI-346 and HI 445, having the specific structure shownin Formula II, where X is Br (HI-346) or Cl (HI-445).

[0020] The CHET compounds and compositions useful in the inventionexhibit:

[0021] 1. very low cytotoxicity;

[0022] 2. very high potency; and

[0023] 3. potent activity against at least one clinically observed drugresistant mutant strain.

[0024] Specific compounds and methods of the invention are describedmore fully in the Detailed Description and in the Examples below.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Definitions

[0026] When used herein, the following terms have the indicatedmeanings:

[0027] “NNI” means non-nucleoside inhibitor. In the context of theinvention, non-nucleoside inhibitors of HIV reverse transcriptase (RT)are defined.

[0028] “Mutant HIV” means a strain of HIV having one or more mutated oraltered amino acids as compared with wild type.

[0029] “Multi-Drug Resistant HIV” means one or more HIV strain which isresistant to treatment with one or more chemotherapeutic agent.

[0030] “Therapeutically effective amount” is a dose which provides sometherapeutic benefit on administration, including, in the context of theinvention, reduced viral activity or viral load in a patient, and alsoincluding inhibition of viral RT activity and/or replication of virus.

[0031] Compounds of the Present Invention

[0032] Compounds of the present invention arecyclohexenyl-ethyl-thiourea (CHET) compounds useful as non-nucleosideinhibitors of RT having the formula I:

[0033] The cyclohexenyl may be substituted or unsubstituted (R_(n)), forexample, R can be H, halogen, (C₁-C₁₂) alkyl or alkoxy, amino, cyano,nitro, hydroxy, and the like. The value of n can be 0 to 6.

[0034] R₁ is a cyclic moiety, which may be substituted or not (X), suchas phenyl, pyridyl, pioeridinyl, piperonyl, morphoryl, furyl, and thelike, and can be, for example, cyclo(C₃-C₁₂) alkyl, cyclo(C₃-C₁₂)alkenyl, isothiazolyl, tetrazolyl, triazolyl, pyridyl, imidazolyl,phenyl, napthyl, benzoxazolyl, benzimidazolyl, thiazolyl, oxazolyl,benzothiazolyl, pyrazinyl, pyridazinyl, thiadiazolyl, benzotriazolyl,pyrolyl, indolyl, benzothienyl, thienyl, benzofuryl, quinolyl,isoquinolyl, pyrazolyl, and the like.

[0035] In one preferred embodiment, R₁ is pyridyl, optionallysubstituted (X) with one or more substituents, for example, with analkyl, alkoxy, halo, or hydroxy group. More preferably, R₁ is pyridylsubstituted with a halogen such as bromine or chlorine. Exemplarycompound of the invention areN-[2-(1-cyclohexenyl)ethyl-N-[2-(5-bromopyridyl)]-thiourea (HI-346) andN-[2-(1-cyclohexenyl)ethyl-N-[2-(5-chloropyridyl)]-thiourea (HI-445).

[0036] The compounds of the invention preferably bind to a specificallosteric site of HIV-RT near the polymerase site and interfere withreverse transcription, for example, by altering either the conformationor mobility of RT.

[0037] Acid salts

[0038] The compounds of the invention may also be in the form ofpharmaceutically acceptable acid addition salts. Pharmaceuticallyacceptable acid addition salts are formed with organic and inorganicacids. As used herein, the compounds of the invention include acid saltsthereof.

[0039] Examples of suitable acids for salt formation are hydrochloric,sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,gluconic, fumaric, succinic, asorbic, maleic, methanesulfonic, and thelike. The salts are prepared by contacting the free base form with asufficient amount of the desired acid to produce either a mono or di,etc. salt in the conventional manner. The free base forms may beregenerated by treating the salt form with a base. For example, dilutesolutions of aqueous base may be utilized. Dilute aqueous sodiumhydroxide, potassium carbonate, ammonia, and sodium bicarbonatesolutions are suitable for this purpose. The free base forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but the salts are otherwise equivalentto their respective free base forms for purposes of the invention. Useof excess base where R is hydrogen gives the corresponding basic salt.

[0040] Methods of Using the Compounds of the Invention

[0041] The compounds of the invention are useful in methods forinhibiting reverse transcriptase activity of a retrovirus. Retroviralreverse transcriptase is inhibited by contacting RT in vitro or in vivo,with an effective inhibitory amount of a compound of the invention. Thecompounds of the invention also inhibit replication of retrovirus,particularly of HIV, such as HIV-1. Viral replication is inhibited, forexample, by contacting the virus with an effective inhibitory amount ofa compound of the invention.

[0042] The methods of the invention are useful for inhibiting reversetranscriptase and/or replication of multiple strains of HIV, includingmutant strains, and include treating a retroviral infection in asubject, such as an HIV-1 infection, by administering an effectiveinhibitory amount of a compound or a pharmaceutically acceptable acidaddition salt of a compound of the Formula I. The compound or inhibitorof Formula I is preferably administered in combination with apharmaceutically acceptable carrier, and may be combined with specificdelivery agents, including targeting antibodies and/or cytokines. Thecompound or inhibitor of the invention may be administered incombination with other antiviral agents, immunomodulators, antibioticsor vaccines.

[0043] The compounds of Formula I can be administered orally, parentally(including subcutaneous injection, intravenous, intramuscular,intrasternal or infusion techniques), by inhalation spray, topically, byabsorption through a mucous membrane, or rectally, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. Pharmaceutical compositionsof the invention can be in the form of suspensions or tablets suitablefor oral administration, nasal sprays, creams, sterile injectablepreparations, such as sterile injectable aqueous or oleagenoussuspensions or suppositories. In one embodiment, the CHET compounds ofthe invention can be applied intravaginally and/or topically, forexample in gel form, for prevention of heterosexual transmission of HIV.

[0044] For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

[0045] For administration by inhalation or aerosol, the compositions canbe prepared according to techniques well-known in the art ofpharmaceutical formulation. The compositions can be prepared assolutions in saline, using benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons or other solubilizing or dispersing agents known in theart.

[0046] For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

[0047] For rectal administration as suppositories, the compositions canbe prepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

[0048] Dosage levels of approximately 0.02 to approximately 10.0 gramsof a compound of the invention per day are useful in the treatment orprevention of retroviral infection, such as HIV infection, AIDS orAIDS-related complex (ARC), with oral doses 2 to 5 times higher. Forexample, HIV infection can be treated by administration of from about0.1 to about 100 milligrams of compound per kilogram of body weight fromone to four times per day. In one embodiment, dosages of about 100 toabout 400 milligrams of compound are administered orally every six hoursto a subject. The specific dosage level and frequency for any particularsubject will be varied and will depend upon a variety of factors,including the activity of the specific compound the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, and diet of the subject, mode of administration, rate ofexcretion, drug combination, and severity of the particular condition.

[0049] The compound of Formula I can be administered in combination withother agents useful in the treatment of HIV infection, AIDS or ARC. Forexample, the compound of the invention can be administered incombination with effective amounts of an antiviral, immunomodulator,anti-infective, or vaccine. The compound of the invention can beadministered prior to, during, or after a period of actual or potentialexposure to retrovirus, such as HIV.

[0050] Conjugation to a Targeting Moiety

[0051] The compound of the invention can be targeted for specificdelivery to the cells to be treated by conjugation of the compounds to atargeting moiety. Targeting moiety useful for conjugation to thecompounds of the invention include antibodies, cytokines, and receptorligands expressed on the cells to be treated.

[0052] The term “conjugate” means a complex formed with two or morecompounds.

[0053] The phrase “targeting moiety” means a compound which serves todeliver the compound of the invention to a specific site for the desiredactivity. Targeting moieties include, for example, molecules whichspecifically bind molecules present on a cell surface. Such targetingmoieties useful in the invention include anti-cell surface antigenantibodies. Cytokines, including interleukins, factors such as epidermalgrowth factor (EGF), and the like, are also specific targeting moietiesknown to bind cells expressing high levels of their receptors.

[0054] Particularly useful targeting moieties for targeting thecompounds of the invention to cells for therapeutic activity includethose ligands that bind antigens or receptors present on virus-infectedcells to be treated. For example, antigens present on T-cells, such asCD48, can be targeted with antibodies. Antibody fragments, includingsingle chain fragments, can also be used. Other such ligand-receptorbinding pairs are known in the scientific literature for targetinganti-viral treatments to target cells. Methods for producing conjugatesof the compounds of the invention and the targeting moieties are known.

[0055] Methods of Making the Compounds of the Invention

[0056] The compounds of the invention may be prepared as shown inSchemes 1 and 2. In general, an appropriate pheneytylamine orpyridylethylamine (R₁—NH₂) is reacted with 1,1′-thiocarbonyl-diimidazole in acetonitrile solvent at ambienttemperature for approximately 12 hours to form a thiocarbonyl reagent.The reaction product is then condensed with a substituted ornon-substituted cyclohexenyl amine in an aprotic solvent such asdimethyl-formamide (DMF) at elevated temperature, such a 100° C., for anextended period of time such as about 15 hours. The desired CHETcompound is purified by column chromatography.

[0057] The CHET compounds of the invention can be synthesized asdescribed above, or by other, known synthetic methods.

EXAMPLES

[0058] The invention may be further clarified by reference to thefollowing Examples, which serve to exemplify the embodiments, and not tolimit the invention in any way.

Example 1 Synthesis and Characterization of Thiourea Inhibitors

[0059] In the present study, we replaced the pyridyl ring of trovirdinewith a cyclohexenyl group that fits well with the Wing 2 region of theNNI binding pocket. The CHET compounds were synthesized as described inScheme 1, in which a thiocarbonyl reagent was prepared fromphenethylamine or pyridylethylamine and 1,1′-thiocarbonyl-diimidazole inacetonitrile solvent at room temperature for 12 hours, and condensedwith the appropriate 2-amino compounds in dimethyl formamide (DMF) at100° C. for 15 hours. After work up, the derivatives were purified bycolumn chromatography. Trovirdine was synthesized according to theliterature procedure.

[0060] Characterization of Synthesized Compounds:

[0061] Proton and carbon nuclear magnetic resonance spectra wererecorded on a Varian spectrometer using an automatic broad band probe.Unless otherwise noted, all NMR spectra were recorded in CDCl₃ at roomtemperature. The chemical shifts reported are in parts per millionrelative to tetramethyl silane as standard. The multiplicity of thesignals were designated as follows: s, d, dd, t, q, m which correspondsto singlet, doublet, doublet of doublet, triplet, quartetand multipletrespectively. UV spectra were recorded from a Beckmann Model #DU 7400UV/Vis spectrometer using a cell path length of 1 cm. Fourier TransformInfra Red spectra were recorded using an FT-Nicolet model Protege #460instrument. The infra red spectra of the liquid samples were run as neatliquids using KBr discs. Mass spectrum analysis was conducted usingeither a Finnigan MAT 95 instrument or a Hewlett-Packard Matrix AssistedLaser Desorption (MALDI) spectrometer model #G2025A The matrix used inthe latter case was cyano hydoxy cinnamic acid. Melting points weredetermined using a Melt John's apparatus and uncorrected. Elementalanalysis were was performed by Atlantic Microlabs (Norcross, Ga.).Column chromatography was performed using silica gel obtained from theBaker Company. The solvents used for elution varied depending on thecompound and included one of the following: ethyl acetate, methanol,chloroform, hexane, methylene chloride and ether. Characterizataion datafor the synthesized compounds is shown below:

[0062]N-[2-(5-Trifluoromethylpyridinyl)]-N′-[2-(1-Cyclohexenyl)ethyl]thiourea(HI-347) yield 40%; mp: 161-162° C.; UV(MeOH) λ_(max): 208, 256, 276,297 nm; IR(KBr) ν3224, 3178, 3039, 2941, 2875, 2831, 1618, 1598, 1552,1531, 1500, 1434, 1324, 1251, 1188, 1164, 1132, 1078, 1016, 933, 871,827, 767, 719, 601 cm⁻¹; ¹H NMR (CDCl₃) δ11.38 (bs, 1H), 9.38 (bs, 1H),8.39 (s, 1H), 7.86-7.82 (dd, 1H), 7.01-6.98 (d, 1H), 5.62 (s, 1H),3.86-3.80 (q, 2H), 2.37-2.33 (t, 2H), 2.05−2.01 (m, 4H), 1.68-1.56 (m,4H); ¹³C NMR (CDCl₃) δ178.8, 155.2, 143.5, 135.6, 134.4, 124.1, 111.9,44.3, 36.8, 27.7, 25.4, 22.8, 22.4;

[0063] N-[2-(5-Bromopyridinyl)]-N′-[2-(1-Cyclohexenyl)ethyl]thiourea(HI-346) yield 45%; mp: 172-173° C.; UV(MeOH) λ_(max): 208, 275, 306 nm;IR(KBr) ν3214, 3156, 3085, 3039, 2925, 2831, 1594, 1560, 1531, 1473,1309, 1267, 1226, 1180, 1135, 1078, 1002, 918. 862, 821, 700, 505 cm⁻¹;¹H NMR (CDCl₃) δ11.21 (bs, 11H), 9.42 (bs, 1H), 8.16-8.15 (d, 1H),7.73-7.69 (dd, 1H), 6.88-6.85 (d, 1H), 5.59 (s, 1H), 3.84-3.78 (q, 2H),2.35-2.31 (t, 2H), 2.05-1.99 (m, 4H), 1.67-1.55 (m, 4H); ¹³C NMR (CDCl₃)δ178.6, 151.8, 146.3, 141.1, 134.5, 124.0, 113.6, 112.7, 44.1, 36.8,27.7, 25.4, 22.8, 22.4;

[0064] N-[2-(5-Bromopyridinyl)]-N′-[2-(2-Adamantyl)]thiourea (HI-504)yield 49%; mp: 239-240° C.; UV(MeOH) λ_(max): 206, 274, 306 nm; IR(KBr)ν3207, 3151, 3081, 3031, 2906, 2844, 1591, 1556, 1531, 1469, 1332, 1309,1270, 1226, 1193, 1172, 1135, 997, 862, 819, 723, 649 cm⁻¹; ¹H NMR(CDCl₃) δ11.51 (bs, 1H), 9.52 (bs, 1H), 8.25-8.24 (d, 1H), 7.74-7.71(dt, 1H), 6.92-6.89 (d, 1H), 3.48-3.46 (d, 2H), 2.05−2.02 (bs, 3H),1.78-1.63 (m, 12H); ¹³C NMR (CDCl₃) δ179.1, 152.0, 146.3, 141.2, 113.7,112.6, 57.9, 40.5, 36.9, 33.7, 28.2;

[0065] N-[2-(5-Bromopyridinyl)]-N′-[2-(2-Myrtanyl)]thiourea (HI-444)yield 55%; mp: 180-181° C.; UV(MeOH) λ_(max): 208, 256, 275, 304 nm;IR(KBr) ν3160, 3029, 2973, 2902, 2854, 1592, 1560, 1531, 1461, 1365,1311, 1267, 1228, 1191, 1139, 1091, 1006, 825, 734, 692, 667, 509 cm⁻¹;¹H NMR (CDCl₃) δ11.35 (bs, 1H), 9.74 (bs, 1H), 8.16 (s, 1H), 7.65-7.61(dd, 1H), 6.90-6.87 (d, 1H), 3.70-3.65 (m, 2H), 2.41-2.28 (m, 2H),1.95−85 (m, 5H), 1.56−1.51 (m, 1H), 1.14 (s, 3H), 1.04 (s, 3H),0.89-0.86 (d, 1H); ¹³C NMR (CDCl₃) δ178.7, 152.0, 146.2, 141.0, 113.8,112.5, 51.2, 43.9, 41.2, 40.5, 38.6, 33.2, 27.9, 25.9, 23.2, 19.8;

[0066] N-[2-(5-Chloropyridinyl)]-N′-[2-(1-Cyclohexenyl)ethyl]thiourea(HI-445) yield 51%; mp: 165° C.; UV(MeOH) λ_(max): 206, 273, 305 nm;IR(KBr) ν3216, 3158, 3087, 3033, 2923, 2831, 1598, 1562, 1533, 1475,1340, 1307, 1228, 1166, 1107, 1018, 910, 862, 823, 692, 586, 505 cm ⁻¹;¹H NMR (CDCl₃) λ11.24 (bs, 1H), 9.67 (bs, 1H), 8.07−8.06 (d, 1H),7.61-7.57 (dt, 1H), 6.99-6.96 (d, 1H), 5.60 (s, 1H), 3.85-3.79 (q, 2H),2.35-2.31 (t, 2H), 2.02−2.00 (d, 4H), 1.67−1.57 (m, 4H); ¹³C NMR (CDCl₃)δ178.5, 151.6, 143.9, 138.4, 134.4, 125.0, 124.0, 113.2, 44.0, 36.8,27.7, 25.4, 22.8, 22.3;

[0067] N-[2-(5-Bromopyridinyl)]-N′-[2-(Pyridinyl)]thiourea (HI-142)(Trovirdine) yield 55%; mp: 152-154° C.; UV(MeOH) λ_(max): 208, 273,306, 485 nm; IR(KBr) ν3224, 3156, 3085, 3039, 2931, 1583, 1558, 1531,1465, 1432, 1361, 1319, 1263, 1228, 1166, 1135, 1095, 1012, 885, 825,756, 700, 661, 567, 511 cm⁻¹; ¹H NMR (CDCl₃) δ11.55 (bs, 1H), 9.56 (bs,1H), 8.61−8.60 (d, 1H), 8.08−8.07 (d, 1H), 7.71−7.62 (m, 2H), 7.29-7.18(m, 2H), 6.89-7.86 (d, 1H), 4.24−4.17 (q, 2H), 3.25-3.21 (t, 2H); ¹³CNMR (CDCl₃) δ178.7, 158.6, 151.6, 148.9, 146.2, 140.9, 136.6, 123.6,121.6, 113.5, 112.6, 44.9, 36.6.

Example 2 Modeling of Designed Thiourea Inhibitors to the NNI BindingPocket

[0068] A computer simulation of the binding of the designed thioureainhibitors to the NNI binding pocket was accomplished using a moleculardocking procedure as described in Vig et al., 1998, Bioorg. Med. Chem.6:1789; Mao, et. al. 1998, Bioorg. Med. Chem. Lett. 8:2213. Once thefinal energetically favored docked position of the inhibitor in the NNIbinding pocket was identified, the inhibitor was assigned an interactionscore, from which the inhibition constant (K_(i)) was estimated. Thedocking results indicated that the cyclohexenyl group of the designedcompounds N-[2-(1-cyclohexenyl)ethyl]-N′-[2-(5-bromopyridyl)]-thiourea(HI-346), andN-[2-(1-cyclohexenyl)ethyl]-N′-[2-(5-chloropyridyl)]-thiourea (HI-445)is situated in the Wing 2 region of the NNI binding pocket, providingcontact with RT residues including Y181.

[0069] The cyclohexenyl group is slightly better than the pyridyl groupof trovirdine relative to its hydrophobic interactions with the RTresidues. According to our modeling studies, the cyclohexenyl group ofthe designed lead compoundN-[2-(1-cyclohexenyl)ethyl]-N′-[2-(5-bromopyridyl)]-thiourea (HI-346)makes 94 hydrophobic contacts with the surrounding RT residues,including P95, Y181, L100, V179, and Y188, and translates into a 3.0 logunit gain in the final interaction score.

[0070] Data are shown below in Table 1. Modeling data are shown in Table1, which indicates the interaction scores and calculated K_(i) values ofcyclohexenyl containing thiourea compounds. In the table, thehydrophobic score (a) is the Jain' score function; the Polar score (b)indicates hydrogen bonding; the solvent effect term (c); and entropicterm (d). N.D. indicates not determined. TABLE 1

Hydrophobic Polar Solvent Entropic K_(i) Compound X Score^(a) Score^(b)c d (μM) HI-346 Br 10.2 1.7 −1.8 −3.3 0.16 HI-445 Cl 9.7 1.7 −1.9 −3.20.50 HI-347 CF₃ 8.8 0.2 −1.6 −3.3 63 Trovirdine 10.2 0.5 −1.2 −3.3 0.63

[0071] The pyridyl group of the reference compound trovirdine bound tothe same region of RT would make 81 contacts with surrounding residues,resulting in a 2.7 log unit gain in the final interaction score. Thealicyclic cyclohexenyl group contains more ring hydrogens than theheterocyclic pyridyl ring and therefore has more hydrogen atom-mediatedcontacts and fewer carbon atom-mediated contacts with RT residues thanthe latter.

[0072] Our composite binding pocket also indicated a region in Wing 1which would be compatible with polar atoms; this region corresponds tothe predicted location of the bound halogen atoms of HI-346 and HI-445.The bromine atom of HI-346 makes 21 contacts with 7 RT residuesincluding H235, L234, and V106, because of its large van der Waalradius. The estimated values for the hydrophobic score function in logunits were 10.2 for HI-346, and 9.7 for HI-445, whereas the estimatedvalues for the polar score function in log units were 1.7 for HI-346 aswell as HI-445. The estimated K_(i) values were 0.16 μM for HI-346 and0.50 μM for HI-445.

Example 3 Antiviral Activity of Substituted Thiourea Compounds

[0073] Purified RT Assays for Anti-HIV Activity

[0074] The synthesized compounds were tested for RT inhibitory activity(IC₅₀[rRT]) against purified recombinant HIV RT using the cell-freeQuan-T-RT system (Amersham, Arlington Heights, Ill.), which utilizes thescintillation proximity assay principle as decribed in Bosworth, et al.,1989, Nature 341:167-168. In the assay, a DNA/RNA template is bound toSPA beads via a biotin/strepavidin linkage. The primer DNA is a 16-meroligo(T) which has been annealed to a poly(A) template. Theprimer/template is bound to a strepavidin-coated SPA bead.

[0075]³H-TTP is incorporated into the primer by reverse transcription.In brief, ³H-TTP, at a final concentration of 0.5 μCi/sample, wasdiluted in RT assay buffer (49.5 mM Tris-Cl, pH 8.0, 80 mM KCl, 10 MmMgCl₂, 10 mM DTT, 2.5 mM EGTA, 0.05% Nonidet-P-40), and added toannealed DNA/RNA bound to SPA beads. The compound being tested was addedto the reaction mixture at 0.001 μM-100 μM concentrations. Addition of10 mU of recombinant HIV RT and incubation at 37° C. for 1 hour resultedin the extension of the primer by incorporation of ³H-TTP. The reactionwas stopped by addition of 0.2 ml of 120 mM EDTA. The samples werecounted in an open window using a Beckman LS 7600 instrument and IC₅₀values were calculated by comparing the measurements to untreatedsamples.

[0076] In addition, the anti-HIV activity of the compounds was measuredby determining their ability to inhibit the replication of the HIV-1strains HTLVIIIB, RT-MDR, A17, and A17 variant in peripheral bloodmononuclear cells (PBMC) from healthy volunteer donors, using the methoddescribed in Uckun et. al., 1998, Antimicrobial Agents and Chemotherapy42:383.

[0077] Normal human peripheral blood mononuclear cells (PBMNC) fromHIV-negative donors were cultured 72 hours in RPMI 1640 supplementedwith 20% (v/v) heat-inactivated fetal bovine serum (FBS), 3%interleukin-2,2 mM L-glutairine, 25 mM HEPES, 2 μL, NAHCO, 50 mg/mLgentamicin, and 4 μg/mL phytohemagglutinin prior to exposure to HIV-1 orother HIV strain at a multiplicity of infection (MOI) of 0.1 during aone-hour adsorption period at 37° C. in a humidified 5% CO2 atmosphere.Subsequently, cells were cultured in 96-well microplates (100 μL/well;2×10⁶ cells/mL, triplicate wells) in the presence of various inhibitorconcentrations. Aliquots of culture supernatants were removed from thewells on the 7^(th) day after infection for p24 antigen p24 enzymeimmunoassays (EIA), as previously described in Erice et al., 1993,Antimicrob. Ag. Chemotherapy 37:385-838. The applied p24 EIA was theunmodified kinetic assay commercially available from CoulterCorporation/Immunotech, Inc. (Westbrook, Me.). Percent inhibition ofviral replication was calculated by comparing the p24 values from thetest substance-treated infected cells with p24 values from untreatedinfected cells (i.e, virus controls).

[0078] A microculture tetrazolium Assay (MTA), using2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazoliumhydroxide (XTT), was performed to evaluate the cytotoxicity of thecompounds, using the methods described, for example, in Uckun et. al.,1998, Antimicrobial Agents and Chemotherapy 42:383; and Mao et. al.,1998, Bioorg. Med. Chem. Lett.8:2213. Data are reported as CC₅₀ (μM).

[0079] Activity Against Drug-Resistant HIV Strains

[0080] The activity of the CHET compounds, HI-445 and HI-346, was testedagainst drug sensitive strains (HTLV IIIB), NNI-resistant strains (A17and A17 variant), as well as multidrug resistant HIV-1 strains (RT-MDR),using the method described in Ucken et al. 1998, Antimicrobial Agentsand Chemotherapy 42:383. RT-MDR was obtained through the AIDS Researchand Reference Reagent Program, from Dr. Bendan Larder, and is describedin Larder et al., 1993, Nature, 365, 451-453.

[0081] Data are presented in Table 2 as the IC₅₀ values for inhibitionof HIV p24 antigen production in PBMC (concentration at which thecompound inhibits p24 production by 50%). Both CHET compounds, HI-346and HI-445, were more effective than trovirdine, and more effective thanas the control NNI compounds nevirapine and delavirdine, in inhibitingrecombinant RT. Furthermore, both CHET compounds were slightly moreeffective than trovirdine, and the control anti-HIV compoundsnevirapine, delavirdine, MKC-442, and AZT, in inhibiting the replicationof the NNI-sensitive/AZT-sensitive HIV-1 strain HTLVIIIB (see Table 2).TABLE 2

IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ A17 CC₅₀ rRT HTLV_(m)B RT-MDR A17 variant MTACompound R₂ X (μM) (μM) (μM) (μM) (μM) (μM) Trovirdine Pyridyl Br 0.80.007 0.020 0.500 >100 >100 HI-346 Cyclohexenyl Br 0.4 0.003 0.001 ND18.7 >100 HI-445 Cyclohexenyl Cl 0.5 0.003 0.001 0.068 30.0 >100 HI-347Cyclohexenyl CF₃ 4.0 0.079 0.038 0.300 >100 >100 HI-504 Adamentyl*Br >100 ND ND ND ND ND HI-444 Myrtanyl* Br >100 ND ND ND ND NDNevirapine — — 23 0.034 5.0 >100 >100 10.5 Delavirdine — — 1.5 0.009 0.450.0 >100 3.6 MKC-442 — — 0.8 0.004 0.3 ND ND >100 AZT — — — 0.004 0.20.006 0.004 >100

[0082] Unlike CHET compounds HI-346 and HI-445, the control cyclohexenylcontaining thiourea compoundN-[2-(1-cyclohexenyl)ethyl]-N′-[2-(5-trifluoromethylpyridyl)]-thiourea(HI-347), for which we had estimated a relatively poor K_(i) value of 63μM, was significanly less potent than trovirdine in both RT inhibitionassays and HIV replication assays. Replacement of the pyridyl ring oftrovirdine with the alicyclic substituents adamantyl or cis-myrtanyl(instead of cyclohexenyl) (R₂) resulted in complete loss of RTinhibitory function (Table 2). Thus, the cyclohexenyl moiety (R₂) aswell as the substitution moiety (x) play critical roles in the anti-HIVactivity of HI-346 and HI-445.

[0083] Notably, both HI-346 and HI-445 were 3-times more effectiveagainst the multidrug resistant HIV-1 strain RT-MDR (V106A mutation andadditional mutations involving the RT residues 74V, 41L, and 215Y) thanthey were against HTLVIIIB. The activity of the lead compound HI-346against RT-MDR was substantially more potent than the activities ofother anti-HIV agents tested. The ranking order of potency againstRT-MDR was: HI-346 (IC₅₀=1 nM)=HI-445 (IC₅₀=1 nM)>trovirdine (IC₅₀=20nM)>AZT (IC₅₀=200 nM)>MKC-442 (IC₅₀=300 nM)>delavirdine (IC₅₀=400nM)>nevirapine (IC₅₀=5000 nM). HI-346 and HI-445 were 20-times morepotent than trovirdine, 200-times more potent than AZT, 300-times morepotent than MKC-442, 400-times more potent than delavirdine, and5000-times more potent than nevirapine against the multi-drug resistantRT-MDR strain.

[0084] HI-445 was also tested against the RTY181C mutant A17 strain andfound to be >7-fold more effective than trovirdine, and >1,400-fold moreeffective than nevirapine or delavirdine. Similarly, both HI-346 andHI-445 were more effective than trovirdine, nevirapine, and delavirdineagainst the trovirdine-resistant A17 variant with both Y181C and K103Nmutations in RT. Neither compound exhibited significant toxicity ateffective concentrations. These findings establish thecyclohexenyl-containing thiourea compounds, and particularly, HI-346 andHI-445, as potent NNI useful against sensitive as well asmultidrug-resistant strains of HIV-1.

[0085] All publications, patents, and patent documents described hereinare incorporated by reference as if fully set forth. The inventiondescribed herein may be modified to include alternative embodiments. Allsuch obvious alternatives are within the spirit and scope of theinvention, as claimed below.

We claim:
 1. A method for treating drug-resistant HIV in a subjectcomprising administering to said subject an effective amount of at leastone compound of the formula

wherein n is 0 to 6; R is H, halogen, (C₁-C₁₂) alkyl, (C₁-C₁₂) alkoxy,amino, cyano, nitro, or hydroxy; and R₁ comprises cyclo(C₃-C₁₂) alkyl,cyclo(C₃-C₁₂) alkenyl, isothiazolyl, tetrazolyl, triazolyl, pyridyl,imidazolyl, phenyl, napthyl, benzoxazolyl, benzimidazolyl, thiazolyl,oxazolyl, benzothiazolyl, pyrazinyl, pyridazinyl, thiadiazolyl,benzotriazolyl, pyrolyl, indolyl, benzothienyl, thienyl, benzofuryl,quinolyl, isoquinolyl, or pyrazolyl; or a pharmaceutically acceptableaddition salt thereof:
 2. The method of claim 1, wherein R₁ issubstituted with one or more of (C₁-C₃)alkyl, (C₁-C₃)alkoxy, halo, orhydroxy.
 3. The method of claim 1, wherein R₁ is pyridyl.
 4. The methodof claim 1, wherein R₁ is pyridyl substituted with halogen.
 5. Themethod of claim 1, wherein R₁ is pyridyl substituted with bromine orchlorine.
 6. The method of claim 1, wherein n is 1 and R is halogen. 7.The method of claim 1, wherein n is
 0. 8. The method of claim 1, whereinthe compound is: N-[2-(1-cyclohexenyl) ethyl]-N¹-[2-(5-bromopyridyl)]-thiourea (HI-346); N-[2-(1-cyclohexenyl) ethyl]-N¹-[2-(5-chloropyridyl)]-thiourea (HI-445); or a pharmaceuticallyacceptable addition salt thereof.